The overarching goal of Project 2 within this multidisciplinary POI Program Project is the development of HIV- 1 entry inhibitors that target the Env spike before the virus encounters the host cell by employing peptide and protein science strategies to design viral entry antagonists. In addition, the derived peptide antagonists, as well as the peptidomimetics emerging from the parent peptides, will be utilized as mechanistic probes to define the vulnerabilities ofthe HlV-1 Env complex to deactivation. Recently, we have discovered a family of peptide triazoles that target a conserved footprint, on gp 120, which overlaps significantly with the CD4 binding site. These inhibitors bind gp120 with nanomolar affinity and block interactions at the sites responsible for binding with both of the cellular receptors required for viral entry. A combination of sequence minimization, non-natural amino acid substitutions, nuclear magnetic resonance and computational simulations have revealed a core pharmacophore in the peptide triazoles that consists of three amino acid residues. Biophysical and functional studies suggest that the inhibitors stabilize a partially structured state of gp 120 and, through an allosteric mechanism, disrupt the conformational transitions of Env complex required to mediate viral entry. In addition, we recently discovered that peptide triazoles, likely by conformational effects, cause gp 120 shedding in the absence of host cells and consequent irreversible inactivation of virus. Further, and of considerable interest, peptide triazole variants containing a free sulfiiydryl group disrupt the structural integrity of HIV-1 envelope membrane, leading to leakage of the intra-viral capsid protein p24. Going forward, these results will be employed to design and validate peptidomimetic and small molecule Env antagonists that inactivate virus. To achieve the overall goals of this Program Project, three Specific Aims will be pursued by Project 2: [1] design, synthesize and develop structure-simplified peptide and peptidomimetic antagonists of HIV-1 envelope and host cell receptor interactions; [2] map the gpl20 binding site for the peptide and peptidomimetic antagonists that define the conformational transitions of gp 120 triggered by peptide triazoles; and [3] determine the mechanistic effects of peptide triazole and peptidomimetic inhibitors on Env protein and virus particle structure and function, and from this understanding, delineate vulnerabilities of the virus Env to antagonism and inactivation. The peptide-based antagonists developed by Project 2 will contribute substantially to the overall effort of this Program Project to understand structural mechanisms of the Env protein entry machine, which in turn holds the promise of providing new tactics for both AIDS prevention and intervention.